Dentin matrix protein 1, a target molecule for Cbfa1 in bone, is a unique bone marker gene.

Dentin matrix protein 1 (Dmp1), a phosphoprotein highly linked to dentin formation, has also been reported to be expressed in the skeleton. However, the role of Dmp1 in skeletal tissues remains unclear. To clarify the role of Dmp1 in bone formation, we characterized the expression profile of Dmp1 in bone and cartilage and examined whether Dmp1 expression was regulated by core-binding factor a1 (Cbfa1). Studies of fetal rat calvarial (FRC) cell cultures showed that the expression of Dmp1 was associated closely with "bone nodule" formation and mineralization in vitro. In situ hybridization studies were performed to examine the spatial and temporal expression patterns of Dmp1 during development in mouse embryos from 12.5 day postcoitus (dpc) to 8 weeks postnatal; these studies showed that Dmp1 first appeared in hypertrophic cartilage cells, followed by osteoblasts, and later was expressed strongly in osteocytes. The expression profiles of Cbfa1 and Dmp1 overlapped in both cartilage and bone during development, with Cbfa1 preceding Dmp1. Examination of Dmp1 expression in Cbfa1-/- mice revealed that Dmp1 was absent in the developing bones of Cbfa1-null mice, whereas there was essentially no change in Dmp1 expression in the arrested tooth bud. Transient transfection studies showed forced expression of Dmp1 under the control of Cbfa1 and gel shift data indicated the presence of a functional osteocalcin-specific element (OSE)-2 response element in the Dmp1 proximal promoter region. However, in vitro promoter studies suggested that regulation of Dmp1 by Cbfa1 was not mediated by direct binding of Cbfa1 to this site and may be through indirect mechanisms. These studies highlight Dmp1 as a unique marker gene for osteoblastic differentiation. The close association of Dmp1 and Cbfa1 in the developing skeleton suggests that Dmp1 may play an important role in bone formation.     

Possible roles of Runx1 and Sox9 in incipient intramembranous ossification.

We evaluated the detailed expression patterns of Runx1 and Sox9 in various types of bone formation, and determined whether Runx1 expression was affected by Runx2 deficiency and Runx2 expression by Runx1 deficiency. Our results indicate that both Runx1 and Sox9 are intensely expressed in the future osteogenic cell compartment and in cartilage. The pattern of Runx1 and Sox9 expression suggests that both genes could potentially be involved in incipient intramembranous bone formation during craniofacial development. INTRODUCTION: Runx1, a gene essential for hematopoiesis, contains RUNX binding sites in its promoter region, suggesting possible cross-regulation with Runx2 and potential regulatory roles in bone development. On the other hand, Sox9 is essential for chondrogenesis, and haploinsufficiency of Sox9 leads to premature ossification of the skeletal system. In this study, we studied the possible roles of Runx1 and Sox9 in bone development. MATERIALS AND METHODS: Runx1, Runx2/Osf2, and Sox9 expression was evaluated by in situ hybridization in the growing craniofacial bones of embryonic day (E)12-16 mice and in the endochondral bone-forming regions of embryonic and postnatal long bones. In addition, we evaluated Runx2/Osf2 expression in the growing face of Runx1 knockout mice at E12.5 and Runx1 expression in Runx2 knockout mice at E14.5. RESULTS: Runx1 and Sox9 were expressed in cartilage, and the regions of expression expanded to the neighboring Runx2-expressing osteogenic regions. Expression of both Runx1 and Sox9 was markedly downregulated on ossification. Runx1 and Sox9 expression was absent in the regions of endochondral bone formation and in actively modeling or remodeling bone tissues in the long bones as well as in ossified craniofacial bones. Runx2 expression was not affected by gene disruption of Runx1, whereas the expression domains of Runx1 were extended in Runx2(-/-) mice compared with wildtype mice. CONCLUSIONS: Runx1 and Sox9 are specifically expressed in the osteogenic cell compartments in the craniofacial bones and the bone collar of long bones, and this expression is downregulated on terminal differentiation of osteoblasts. Our results suggest that Runx1 may play a role in incipient intramembranous bone formation.     

Runx1/AML1/Cbfa2 mediates onset of mesenchymal cell differentiation toward chondrogenesis.

Runx proteins mediate skeletal development. We studied the regulation of Runx1 during chondrocyte differentiation by real-time RT-PCR and its function during chondrogenesis using overexpression and RNA interference. Runx1 induces mesenchymal stem cell commitment to the early stages of chondrogenesis. INTRODUCTION: Runx1 and Runx2 are co-expressed in limb bud cell condensations that undergo both cartilage and bone differentiation during murine development. However, the cooperative and/or compensatory effects these factors exert on skeletal formation have yet to be elucidated. MATERIALS AND METHODS: Runx1/Cbfa2 and Runx2/Cbfa1 were examined at different stages of embryonic development by immunohistochemistry. In vitro studies used mouse embryonic limb bud cells and assessed Runx expressions by immunohistochemistry and real-time RT-PCR in the presence and absence of TGFbeta and BMP2. Runx1 was overexpressed in mesenchymal cell progenitors using retroviral infection. RESULTS: Immunohistochemistry showed that Runx1 and Runx2 are co-expressed in undifferentiated mesenchyme, had similar levels in chondrocytes undergoing transition from proliferation to hypertrophy, and that there was primarily Runx2 expression in hypertrophic chondrocytes. Overall, the expression of Runx1 remained significantly higher than Runx2 mRNA levels during early limb bud cell maturation. Treatment of limb bud micromass cultures with BMP2 resulted in early induction of both Runx1 and Runx2. However, upregulation of Runx2 by BMP2 was sustained, whereas Runx1 decreased in later time-points when type X collagen was induced. Although TGFbeta potently inhibits Runx2 and type X collagen, it induces type II collagen mRNA and mildly but significantly inhibits Runx1 isoforms in the early stages of chondrogenesis. Virus-mediated overexpression of Runx1 in mouse embryonic mesenchymal cells resulted in a potent induction of the early chondrocyte differentiation markers but not the hypertrophy marker, type X collagen. Knockdown or Runx1 potently inhibits type II collagen, alkaline phosphatase, and Runx2 and has a late inhibitory effect on type X collagen. CONCLUSION: These findings show a distinct and sustained role for Runx proteins in chondrogenesis and subsequent chondrocyte maturation. Runx1 is highly expressed during chondrogenesis in comparison with Runx2, and Runx1 gain of functions stimulated this process. Thus, the Runx genes are uniquely expressed and have distinct roles during skeletal development.     

PTH Receptor Signaling in Osteoblasts Regulates Endochondral Vascularization in Maintenance of Postnatal Growth Plate

Longitudinal growth of postnatal bone requires precise control of growth plate cartilage chondrocytes and subsequent osteogenesis and bone formation. Little is known about the role of angiogenesis and bone remodeling in maintenance of cartilaginous growth plate. Parathyroid hormone (PTH) stimulates bone remodeling by activating PTH receptor (PTH1R). Mice with conditional deletion of PTH1R in osteoblasts showed disrupted trabecular bone formation. The mice also exhibited postnatal growth retardation with profound defects in growth plate cartilage, ascribable predominantly to a decrease in number of hypertrophic chondrocytes, resulting in premature fusion of the growth plate and shortened long bones. Further characterization of hypertrophic zone and primary spongiosa revealed that endochondral angiogenesis and vascular invasion of the cartilage were impaired, which was associated with aberrant chondrocyte maturation and cartilage development. These studies reveal that PTH1R signaling in osteoblasts regulates cartilaginous growth plate for postnatal growth of bone. © 2014 American Society for Bone and Mineral Research.     

Role of IGFBP2, IGF-I and IGF-II in regulating long bone growth

The IGF axis is important for long bone development, homeostasis and disease. The activities of IGF-I and IGF-II are regulated by IGF binding proteins (IGFBPs). IGF-I and IGFBP2 are co-expressed in dynamic fashions in the developing long bones of the chick wing, and we have found that IGF-II is present in the cartilage model and surrounding perichondrium, proliferative and hypertrophic chondrocytes and developing periosteum. To gain insight into endogenous roles of IGF-I, IGF-II and IGFBP2 in long bone development, we have overexpressed IGFBP2 in the developing skeletal elements of the embryonic chick wing in vivo, using an RCAS retroviral vector. IGFBP2 overexpression led to an obvious shortening of the long bones of the wing. We have investigated, at the cellular and molecular levels, the mechanism of action whereby IGFBP2 overexpression impairs long bone development in vivo. At an early stage, IGFBP2 excess dramatically inhibits proliferation by the chondrocytes of the cartilage models that prefigure the developing long bones. Later, IGFBP2 excess also reduces proliferation of the maturing chondrocytes and attenuates proliferation by the perichondrium/developing periosteum. IGFBP2 excess does not affect morphological or molecular indicators of chondrocyte maturation, osteoblast differentiation or cell/matrix turnover, such as expression of Ihh, PTHrP, type X collagen and osteopontin, or distribution and relative abundance of putative clast cells. We also have found that IGFBP2 blocks the ability of IGF-I and IGF-II to promote proliferation and matrix synthesis by wing chondrocytes in vitro. Together, our results suggest that the mechanism of action whereby IGFBP2 excess impairs long bone development is to inhibit IGF-mediated proliferation and matrix synthesis by the cartilage model; reduce the proliferation and progression to hypertrophy by the maturing chondrocytes; and attenuate proliferation and formation of the periosteal bony collar. These actions retard the growth and longitudinal expansion of the developing long bones, resulting in shortened wing skeletal elements. Our results emphasize the importance of a balance of IGF/IGFBP2 action at several stages during normal long bone development.     

Role of IGFBP2, IGF-I and IGF-II in regulating long bone growth

The IGF axis is important for long bone development, homeostasis and disease. The activities of IGF-I and IGF-II are regulated by IGF binding proteins (IGFBPs). IGF-I and IGFBP2 are co-expressed in dynamic fashions in the developing long bones of the chick wing, and we have found that IGF-II is present in the cartilage model and surrounding perichondrium, proliferative and hypertrophic chondrocytes and developing periosteum. To gain insight into endogenous roles of IGF-I, IGF-II and IGFBP2 in long bone development, we have overexpressed IGFBP2 in the developing skeletal elements of the embryonic chick wing in vivo, using an RCAS retroviral vector. IGFBP2 overexpression led to an obvious shortening of the long bones of the wing. We have investigated, at the cellular and molecular levels, the mechanism of action whereby IGFBP2 overexpression impairs long bone development in vivo. At an early stage, IGFBP2 excess dramatically inhibits proliferation by the chondrocytes of the cartilage models that prefigure the developing long bones. Later, IGFBP2 excess also reduces proliferation of the maturing chondrocytes and attenuates proliferation by the perichondrium/developing periosteum. IGFBP2 excess does not affect morphological or molecular indicators of chondrocyte maturation, osteoblast differentiation or cell/matrix turnover, such as expression of Ihh, PTHrP, type X collagen and osteopontin, or distribution and relative abundance of putative clast cells. We also have found that IGFBP2 blocks the ability of IGF-I and IGF-II to promote proliferation and matrix synthesis by wing chondrocytes in vitro. Together, our results suggest that the mechanism of action whereby IGFBP2 excess impairs long bone development is to inhibit IGF-mediated proliferation and matrix synthesis by the cartilage model; reduce the proliferation and progression to hypertrophy by the maturing chondrocytes; and attenuate proliferation and formation of the periosteal bony collar. These actions retard the growth and longitudinal expansion of the developing long bones, resulting in shortened wing skeletal elements. Our results emphasize the importance of a balance of IGF/IGFBP2 action at several stages during normal long bone development.     

Deficiency of insulin receptor substrate-1 impairs skeletal growth through early closure of epiphyseal cartilage.

Morphological analyses in and around the epiphyseal cartilage of mice deficient in insulin receptor substrate-1 (IRS-1) showed IRS-1 signaling to be important for skeletal growth by preventing early closure of the epiphyseal cartilage and maintaining the subsequent bone turnover at the primary spongiosa. Introduction: IRS-1 is an essential molecule for intracellular signaling by IGF-I and insulin, both of which are potent anabolic regulators of cartilage and bone metabolism. To clarify the role of IRS-1 signaling in the skeletal growth, morphological analyses were performed in and around the epiphyseal cartilage of mice deficient in IRS-1 (IRS-1(-/-)), whose limbs and trunk were 20-30% shorter than wildtype (WT) mice. MATERIALS AND METHODS: The epiphyseal cartilage and the primary spongiosa at proximal tibias of homozygous IRS-1(-/-) and WT male littermates were compared using histological, immunohistochemical, enzyme cytohistochemical, ultrastructural, and bone histomorphometrical analyses. RESULTS: In and around the WT epiphyseal cartilage, IRS-1 and insulin-like growth factor (IGF)-1 receptors were widely expressed, whereas IRS-2 was weakly localized in bone cells. Chronological observation revealed that height of the proliferative zone and the size of hypertrophic chondrocytes were decreased in WT mice as a function of age, and these decreases were accelerated in the IRS-1 (-/-) cartilage, whose findings at 12 weeks were similar to those of WT at 24 weeks. In the IRS-1(-/-) cartilage, proliferating chondrocytes with positive proliferating cell nuclear antigen (PCNA) or parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor immunostaining had almost disappeared by 12 weeks. Contrarily, TUNEL+ apoptotic cells were increased in the hypertrophic zone, at the bottom of which most of the chondrocytes were surrounded by the calcified matrix, suggesting the closure of the cartilage. In the primary spongiosa, bone volume, alkaline phosphatase (ALP)+ osteoblasts, TRACP+ osteoclasts, and the osteopontin-positive cement line were markedly decreased. Bone histomorphometrical parameters for both bone formation and resorption were significantly lower in IRS-1(-/-) mice, indicating the suppression of bone turnover. CONCLUSION: The IRS-1(-/-) epiphyseal cartilage exhibited insufficient proliferation of chondrocytes, calcification of hypertrophic chondrocytes, acceleration of apoptosis, and early closure of the growth plate. Thus, the data strongly suggest that IRS-1 signaling is important for the skeletal growth by preventing early closure of the epiphyseal cartilage and by maintaining the subsequent bone turnover at the primary spongiosa.     

Developmental and TGF-beta-mediated regulation of Ank mRNA expression in cartilage and bone

OBJECTIVES: Ank encodes a transmembrane protein that is involved in pyrophosphate (PPi) transport and mutations in the Ank gene have been associated with pathological mineralization in cartilage and bone. To understand how Ank works in normal skeletal development it is also important to know which cells within the developing skeleton express Ank. To this end, we examined the expression pattern of Ank mRNA during mouse embryonic development as well as in mouse hind limb joints with emphasis on the period when articular cartilage forms. Since it was previously shown that TGF-beta regulates PPi transport in cells in culture, we also tested the hypothesis that TGF-beta regulates Ank expression. METHODS: The localization of Ank mRNA was determined by radioactive in situ hybridization in E15.5 and E17.5 mouse embryos as well as in 1 and 3 week post-natal mice. Ank expression was compared to that of other cartilage markers. In situ hybridization and semi-quantitative RT-PCR were used to determine the effects of TGF-beta on Ank expression in metatarsal organ cultures. RESULTS: Ank expression was detected at high levels at sites of both endochondral and intramembranous bone development. In endochondral bones, expression was detected in a subset of hypertrophic cells at ossification centers. Expression was also detected in osteogenic/chondrogenic cells of the perichondrium/periosteum lining the metaphysis, an area associated with the formation and extension of the bone collar. High levels of expression were also detected in non-mineralized tissues of the skeletal system including tendons and the superficial layer of the articular cartilage. Treatment with TGF-beta resulted in an approximately four-fold induction of Ank mRNA in prehypertrophic chondrocytes and perichondrium of metatarsal cultures.CONCLUSIONS: The expression pattern of Ank suggests an important role both in inhibiting and regulating mineralization in the developing skeletal system. In addition, TGF-beta1 is able to mediate Ank mRNA expression in chondrocytes suggesting a possible role for TGF-beta and Ank in the regulation of normal mineralization.     

Quantitative histomorphometric analysis of the human growth plate from birth to adolescence

Longitudinal bone growth occurs via the transformation of growth plate cartilage into bone through a series of cell and matrix changes, termed endochondral ossification. In this study, we characterize the development of trabecular bone from growth plate cartilage in the human rib from birth to adolescence. The height of the proliferative and hypertrophic zones within the growth plate and the primary bone spongiosa decreased with increasing age, with the greatest change observed in the first year of postnatal life. Within these zones, an internal rearrangement of tissue structure occurred. The matrix volume fraction (either cartilage or bone) increased with age in each of the zones. A concomitant increase in cartilage septae thickness and bone trabecular thickness was observed. A decrease in cartilage septae number was seen in the proliferative zone and a decrease in bone trabeculae number was also observed in the primary spongiosa. However, no difference in cartilage septae number was noted in the hypertrophic zone, the region at which cartilage is transformed into bone. Together the proliferative and hypertrophic regions of the growth plate and the bone primary spongiosa appear to constitute the active growth region, with concomitant changes observed that result in longitudinal growth. In contrast, bone mineral volume in the secondary spongiosa was stable over the ages examined; however, trabecular architecture underwent consolidation as trabecular number decreased and trabecular thickness increased. The integration of the structural transformation from cartilage to bone is crucial in achieving the dual purposes of longitudinal growth and peak bone mass. The structure developed during childhood will have an important bearing on the response to bone-altering disease in later life.     

Reduction in Gsalpha induces osteogenic differentiation in human mesenchymal stem cells

We hypothesized that a decrease in Gsalpha expression occurs with osteogenic differentiation and that when Gsalpha expression was decreased by antisense oligonucleotides or direct inhibition of protein kinase A there was a concomitant increase in Runx2/Cbfa1. We also investigated the mechanism involved in the change in Runx2/Cbfa1 levels and whether the expression of other genes known to be involved in bone formation was altered. There was a decrease in Gsalpha expression with osteogenic differentiation and antisense oligonucleotides, and protein kinase A inhibition led to increased expression and DNA binding of the osteoblast-specific Runx2/Cbfa1. Additionally, with decreased Gsalpha expression or protein kinase A inhibition, Runx2/Cbfa1 protein was serine phosphorylated and ubiquitinated less. Microarray analysis, after the addition of antisense Gsalpha, showed a more than 10-fold increase in collagen Type I Alpha 2 mRNA (a target of Runx2/Cbfa1). These data show that reduced expression of Gsalpha can induce an osteoblast-like phenotype. The results also indicate a potential pathophysiologic role in patients with heterozygous inactivating mutations in GNAS1, the gene for the alpha chain (Gsalpha) of the heterotrimeric G protein, present in three disorders with ectopic intramembranous bone: Albright's hereditary osteodystrophy, progressive osseous heteroplasia, and osteoma cutis.     

Cartilage abnormalities are associated with abnormal Phex expression and with altered matrix protein and MMP-9 localization in Hyp mice

X-linked hypophosphatemic rickets (HYP) in humans is caused by mutations in the PHEX gene. This gene mutation is also found in Hyp mice, the murine homologue of the human disease. At present, it is unknown why loss of Phex function leads to cartilage abnormalities in Hyp mice. In the present study, we compared in wild-type and Hyp mice Phex protein localization in cartilage of developing long bone as well as localization of skeletal matrix proteins and matrix metalloproteinase-9 (MMP-9). Also compared were chondrocyte apoptosis in the growth plate, mineralization and cartilage remnant retention in the metaphysis, and chondroclast/osteoclast characteristics in the primary spongiosa. Phex protein was detected in proliferating and hypertrophic chondrocytes in growth plate cartilage of wild-type mice, but not in Hyp mice. Hyp mice exhibited a widened and irregular hypertrophic zone in growth plate cartilage showing hypomineralization, increased cartilage remnants from the growth plate in both metaphyseal trabecular and cortical bone, and fewer and smaller chondroclasts/osteoclasts in the primary spongiosa. Increased link protein and C-propeptide of type II procollagen of Hyp mice reflected the increase in chondrocytes and matrix in the cartilaginous growth plate and in bone. In addition, growth plate osteocalcin and bone sialoprotein levels were decreased, while osteonectin was increased, in hypertrophic chondrocytes and cartilage matrix in Hyp mice. MMP-9 in hypertrophic chondrocytes was also reduced in Hyp mice and fewer apoptotic hypertrophic chondrocytes were detected. These findings suggest that Phex may control mineralization and removal of hypertrophic chondrocytes and cartilage matrix in growth plate by regulating the synthesis and deposition of certain bone matrix proteins and proteases such as MMP-9.     

Distribution of type X collagen in tibiotarsi of broiler chickens with vitamin D deficiency

Summary Type X collagen is a significant component of the extracellular matrix of the hypertrophic zone of physeal cartilage, but its precise role in endochondral ossification has not been determined. The concentration of type X collagen increases in physeal cartilage in chicks with vitamin D deficiency. The purpose of our study was to determine whether defective endochondral ossification due to vitamin D deficiency was associated with abnormalities in the distribution of type X collagen in the proximal tibiotarsus of chicks. To accomplish this, we induced vitamin D deficiency in broiler chicks and sequentially evaluated the pattern of type X collagen immunoreactivity in the proximal tibiotarsus using a monoclonal antibody specific for chicken type X collagen. Type X collagen immunoreactivity was present in the matrix of the prehypertrophic zone, hypertrophic zone, cartilage cores of the primary spongiosa, and within the chondrocytes of the prehypertrophic and early hypertrophic zones in vitamin D-deficient and D-replete chicks. However, rachitic chicks exhibited two consistent differences in type X collagen immunoreactivity: hypertrophic chondrocytes in the late hypertrophic zone and primary spongiosa contained intracellular type X collagen; and type X collagen was concentrated into laminated aggregates in the pericellular and territorial matrices in the late hypertrophic zone and primary spongiosa. We conclude from these findings that (1) normal serum concentrations of 1,25(OHD)₂D₃ and 25OHD₃ are not required for type X collagen production; (2) type X collagen production does not decrease in the most mature zones of the physes in chicks with vitamin D deficiency; and (3) newly secreted type X collagen accumulates in the pericellular and territorial matrices of the late hypertrophic zone and primary spongiosa of rachitic chicks, perhaps because it is not readily incorporated into the interterritorial matrix.